MITOCHONDRIAL PROTEOMICS OF AGING

描述(申请人提供)：这项建议的目的是对衰老对小鼠线粒体蛋白质组的影响进行全面的检查。我们选择关注线粒体蛋白质组有两个原因。首先，人们几乎一致认为，线粒体很可能在衰老中发挥重要作用，无论是通过功能变化(例如，能量产生减少或增加凋亡)，还是通过产生ROS，后者反过来破坏导致衰老的分子。其次，利用蛋白质组学的当前技术，线粒体蛋白质组有一个可管理的蛋白质数量(1000到2000个)要研究。 虽然有大量的数据表明线粒体功能随着年龄的增长而恶化，但关于这种现象的分子基础的信息很少。我们假设线粒体蛋白质组的改变很可能在与年龄相关的线粒体功能改变中起主要作用。我们建议通过首次详细研究线粒体蛋白质组的全球表达和翻译后修改作为年龄的函数来检验这一假说，方法是研究从小鼠不同组织中分离的线粒体在其生命周期中的作用，并确定饮食限制(DR)对线粒体蛋白质组中观察到的与年龄相关的变化的影响。这个项目的具体目的是描述AGE和DR对1)线粒体蛋白质组的蛋白质表达，2)线粒体磷酸蛋白质组的磷酸化，3)线粒体蛋白质的氧化修饰，以及4)线粒体功能的影响。这些研究将使用我们专门为检测蛋白质表达、酶翻译后修饰和蛋白质氧化而开发的一系列新的蛋白质组学方法进行。认识到多维层析和凝胶电泳经常可以看到不同的蛋白质，我们设计了允许我们在线粒体蛋白质组研究中使用这两种技术的方法。

DESCRIPTION (provided by applicant): The goal of this proposal is to conduct a comprehensive examination of the effect of aging on the mitochondrial proteome of mice. We have chosen to focus on the mitochondrial proteome for two reasons. First, there is almost unanimous agreement that mitochondria are very likely to play an important role in aging, either through changes in function (e.g., reduced energy generation or increase apoptosis) or through the generation of ROS, which in turn damage molecules leading to aging. Second, the mitochondrial proteome has a manageable number of proteins (1000 to 2000) to study using the current technology in proteomics. Although there are substantial data on the deterioration of mitochondrial function with aging, there is little information on the molecular basis of this phenomenon. We hypothesize that alterations in the mitochondrial proteome have a high probability of playing a major role in the age-related changes in mitochondria function. We proposed to test this hypothesis by performing the first detailed study of global expression and post-translational modifications in mitochondria proteome as a function of age by studying mitochondria isolated from various tissues of mice over their life span and determining the effect of dietary restriction (DR), which retards aging, on the age-related changes observed in the mitochondrial proteome. The Specific Aims of this project are to profile the effects of age and DR on 1) protein expression in the mitochondria proteome, 2) phosphorylation of the mitochondria phosphoproteome, 3) oxidative modification of mitochondria proteins, and 4) mitochondrial function. These studies will be conducted with a series of new proteomics methods we have developed specifically for the examination of protein expression, enzymatic post-translational modifications, and protein oxidation. Recognizing that different proteins are often seen with multidimensional chromatography and gel electrophoresis, we have designed methods that allow us to employ both techniques in our studies of the mitochondrial proteome.